Alcoholic Steatohepatitis (ASH) is a major cause of liver related mortality. Despite its public health significance, there has been limited therapeutic advances for ASH. A principal barrier to acceleration of therapeutics is the availability of a robust preclinical model of ASH which recapitulates human ASH. Such models are needed to better understand the role of specific genes and pathways in human ASH e.g. the PNPLA3 mutation which is associated with severe ASH and test promising compounds for both efficacy and safety. In this UH2/UH3 application, we propose to provide ?proof of concept? that our recently validated diet-induced animal model of NAFLD (DIAMOND) can be leveraged as a model of ASH with specific alcohol feeding protocols and will test the following novel hypothesis: an inbred isogenic crossed C57Bl/6J and 129S1/SvlmJ (B6/S129) mouse strain will develop steatohepatitis that recapitulates the key features of human ASH with alcohol feeding. Furthermore, liver-specific expression of the human mutant I148M PNPLA3 in this mouse will accelerate the development of ASH upon alcohol feeding. As per RFA AA-18-006, the studies will be performed in two phases: UH2 Phase (yrs. 01-02): To demonstrate that alcohol feeding causes steatohepatitis resembling human ASH with respect to histology, markers of liver injury and function, and activation of signaling pathways in an isogenic strain of B6/S129 mice. Alcohol feeding will be done along with chow- or an obesogenic-diet. We will test the effect of the NIAAA alcohol model feeding strategy (to be performed at NIAAA under supervision by Dr. Gao) and an alternate strategy where we will perform single ascending dose (SAD) and multiple ascending dose (MAD) studies that will provide optimized alcohol feeding strategies including amounts given chronically along with binges to allow a phenotype of ASH to develop. We will also test the ability to accelerate development of ASH by liver-specific expression of the human I148M PNPLA3 mutant gene. These data are based on preliminary data indicating that NASH can be accelerated by this maneuver. A minimal requirement for development of ASH (steatohepatitis, increased AST and bilirubin) will be needed to proceed to the UH3 phase. UH3 Phase (yrs. 03-05): To further validate the model, define the course of disease progression and regression by modulation of alcohol intake, and the impact of ASH on the susceptibility to ASH in subsequent generations. The transcriptome, metabolome and microbiome of the model will be related to human data from the AlcHepNet consortium. Also, the effects of alcohol withdrawal after varying durations of exposure will be tested to define the ?off-response?. We will also perform studies to determine the susceptibility of offspring of mice that have been allowed to develop ASH and then recover by withdrawing alcohol. Microbiome analyses will be done at the UCSD site of AlcHepNet. The investigators have the required expertise in ASH and mouse models of NASH and ASH. Together the studies will have a high impact by providing a mouse model of ASH. The project also meets the high-risk high reward criteria for the UH2/UH3 funding mechanism.